JPS61502892A - t-PA compositions and methods of incorporating them into the blood - Google Patents

Abstract

The absorption rate of protein-thrombolytic agents with medicinal properties in the blood is enhanced by administering the protein intramuscularly together with an absorption enhancing agent, e.g. hydroxylamine or a salt thereof.

Description

[Detailed description of the invention] t-PA composition and method for incorporating it into the blood Regarding treatments to minimize damage to the heart muscle during severe pain due to myocardial infarction in pancreatic patients. , especially by improving such procedures so that professionals can directly care for patients. It is about being able to start benefits as early as possible, even before Ru.

When blood clots form within a blood vessel, the body tissues supplied by this blood vessel are at risk. exposed or completely deprived of blood supply. This happens depending on the blood vessels. The threat to a person's life can be small, small, or very large; As mentioned above, i.e. an environment that threatens life of some kind. There is. The formation of a clot within a blood vessel is described as thrombosis. substance that dissolves blood clots The substance is called a thrombolytic substance. Blood clots in the coronary arteries are dissolved, resulting in damage to the heart. Once blood flow is established, it is called reperfusion.

Examples of life-threatening clots that form in arteries include cerebral thrombosis, renal thrombosis, and ophthalmic artery thrombosis. , and most likely have serious coronary artery blood clots. Severe myocardial infarction (coronary heart attack) Approximately 85% to 90% of patients have a blood clot in the coronary artery, which causes blood to flow to the heart muscle. The flow to the meat (heart muscle) and the supply of essential oxygen and other nutrients is inhibited. crown The formation of a blood clot in a blood clot in an artery affects the myocardium (the heart muscle that pumps the heart). organization). The heart muscle deprived of blood supply does not die immediately, but it does die. Begin the process to reach it. Therefore, the degree of injury to the heart muscle depends on the degree of damage to the infarcted area. It is a function of the time during which the blood supply is restricted by clotting or PA occlusion.

Traditionally, the methods used to actually establish reperfusion to the infarcted area have always been limited to the hospital environment. It was carried out at the border or an equivalent place. So-called “pre-hospital” treatment generally involves Keep the patient alive and get the patient into a hospital environment as soon as possible to treat damage to the heart muscle. The aim was to enable treatment to minimize harm.

Some procedures performed in a hospital setting establish reperfusion into the infarcted portion of a patient's heart. Contains certain methods for.

Establishing reperfusion is not recommended unless surgery is clearly indicated as immediately necessary. It was carried out with a procedure that has the effect of passing. These methods include catheterization and thrombolysis. This includes the administration of drugs. Known thrombolytic agents such as streptokinase and urokinase Removal requires intracardiac injection, i.e., using a catheter into the blocked blood vessel. Gradually inject the lysing agent. In recent years, intravenous infusion of streptokinase has become effective. It has been proven that.

More recently, a substance called tissue-type degrading enzyme activator, or t-PA, has been used experimentally. was used. (The New England Journal of Medi cine, March 8, 1984, Vol. 310, No. 10, pp. 609-610).

Unlike other degradative enzyme activators such as streptokinase and urokinase, t-PA exists in only a small amount in the body, but if it is maintained at an appropriate and effective amount. It acts on coagulation, but not on other proteins in the blood.

Biochemical Pharmacology + Volume 33 No. 12, 18 Pages 31 to 1838, “Cardiac thrombosis: Tissue-type enzyme activator (t-FA) f, strong In a 1984 article titled ``Pharmacological Considerations,'' the following concluding remarks were made: Contains a description.

[The selection of pharmacological aids for the induction of cardiac thrombosis is determined primarily by availability.] It was Unfortunately, both streptokinase and urokinase are Circulating fibrin, degrading enzymes, and α2-antiplasmin are reduced, and their release from fibrin is reduced. Accumulation of decomposition products induces a lytic state in the tissue, and all these factors overlap. creating a bleeding condition that can lead to serious bloodshed. Intravenous administration of these drugs It is not very common because the success rate is low, and one of the reasons is that it induces a state of severe tissue lysis. This is because the upper limit of the drug dose is restricted due to the risk of

Advances in recombinant DNA engineering may expand the availability of tissue-based degradative enzyme activators. This possibility is of particular interest due to the coagulation properties of t-PA. Ru.

The potential advantages of t-PA against coronary thrombosis include the safety of high-dose intravenous administration. Completeness and efficiency; effective coagulation and lysis without inducing tissue lysis extensive characterization of each patient's coagulation and fibrinolytic tissue before or during treatment; Can be performed immediately without the need for a sex test; no obvious allergic reaction or immunity Changes in response to drug dose can be avoided depending on the composition of epidemics; Because of the ease of minute-to-moment adjustment and the short biological half-life of t-PA, If necessary, fibrinolysis can be terminated immediately as there is no risk of inducing a fibrinolytic state. This includes things such as what you can do.

The point where t-FA administration is expected to contribute is American HeartAssoc. It was discussed in the information committee of the ation meeting and dated November 16, 1983. Reported in an article in the New York Times. The title is ``Cancer used to prevent heart disease.'' "cell proteins." This article considers the injection of t-PA and explains the following. "This protein (t-PA) is found in the arms of patients who have suffered myocardial infarction or heart attack. It can be easily injected into a vein and travels through the blood to dissolve clots. this is water supply It's very similar to the cleaning agent used to clean clogged pipes. ” This article is ``Hope for future applications.'' With the subtitle, many doctors use t-PA for the threat of heart attack. It is reported that he expressed interest in conducting research on Doctors will someday t-P A can be used in first aid rooms and ambulances, and should not cause death or permanent injury. The hope was that it would be possible to stop heart attacks early before they occur. . In this column of ``hope for future application,'' there was also the following sentence: “One of the researchers, Wa Dr. Burton E., of Sinton University, estimates that the patient will someday If you have a vial with you and feel chest pain or other early symptoms of a heart attack, give the medication immediately. I want to be able to shoot.''

In medical terms, a vial is a container for a quantity of liquid medicine or diluent. The bottle has a rubber stopper that can be inserted with the needle of the syringe. A predetermined amount of liquid can be drawn out using a syringe. Intravenous injection In practice, it is difficult to administer the injection yourself. This can lead to severe disability and an upsetting cycle in which patients endure symptoms of myocardial infarction. This is especially true when considering boundaries.

An effective method of self-administration of t-FA has been developed to treat certain patients susceptible to heart disease. If it can be used as soon as symptoms start, it can be used as a thrombolytic agent for t-PA. significantly increases the likelihood of efficacy. And irreversible damage to the heart muscle occurs. It can be used very early, before the patient is hospitalized, and at the same time, before hospitalization or before boarding an ambulance. type of treatment, which can help minimize myocardial damage associated with myocardial infarction. It is directly effective and is the first of its kind.

It is an object of the present invention to provide such a self-administered treatment.

Another object of the present invention is to increase the absorption rate of 1-PA in the blood when injected intramuscularly. There is a particular thing.

The present invention also relates to t-PA preparations and preparations that enhance the absorption of t-FA in blood. It is. The formulation is hydroxylamine hydrochloride in a known emergency auto-injector. Yes, two types of drugs are administered into the muscle tissue after receiving instructions from a doctor over the phone, and once directly. Inject before receiving medical attention.

When t-PA is injected into the blood at a predetermined concentration, t-PA is clot selective. It can be used as a platelet lytic agent, but tests conducted in this case have shown that overall hemolysis It has been found that the concentration can be increased to the point where the condition can occur. by intramuscular injection near the wound caused by insertion or withdrawal of the hypodermic needle. A concentration of t-PA can be injected into the affected area and the area that substantially surrounds the wound. can. As a result, hemolysis occurs at least locally, and the syringe causes bleeding from the wound. It will bleed.

Contrary to expectations, tests revealed that the above-mentioned bleeding did not actually occur.

Second, t-PA is a protein with a large molecular weight. t-PA can be recognized in the blood It was not expected that such amount would be absorbed. The amount of white matter outside the vascular bundle is the amount of protein inside the vascular bundle. It is about 1/1 o of the view. Capillary pores, where protein transport takes place, are protein molecules. Because certain proteins are transported by charge, they are small compared to the size of The above facts are considered to be natural. Large proteins such as t-PA can be injected intramuscularly. It can be recognized when it is injected by injection, that is, when it is injected outside the blood vessel. The question of whether a sufficient amount could be rapidly injected into the bloodstream was a very difficult problem. Application exam Experiments have shown that therapeutically significant amounts of t-PA are rapidly released into the blood after intramuscular injection with t-PA alone. It turns out that crabs are not injected.

Therefore, in actual treatment, t-PA should be absorbed at the same time as the intramuscular injection or at substantially the same time. The enhancer must be injected intramuscularly to reach the concentration necessary for platelet 5 lysis.

Enhances the absorption of low molecular weight substances administered topically, subcutaneously or intramuscularly For example, a solvent such as dimethyl sulfoxide (DMSO) may be used to One must try to increase the flow of blood and lymph to the skeletal muscles.

However, DMSO was not found to be effective as an absorption enhancer for t-PA. Ta.

According to the principles of the present invention, the rate of absorption of t-PA in the blood increases with the t-PA formulation. Used with agents that enhance absorption of FA, preferably hydroxylamine hydrochloride. It is promoted by Preferably absorption enhancers such as hydroxylamine Hydrochloride 'it-PA formulation to form a single mixture for intramuscular injection (i, m, ) as a drug. The intent of the present invention is to inject the individual formulations of t-PA into the same site where they were injected. Injecting absorption enhancers (e.g. U.S. Pat. No. 4,394°863) However, absorption-enhancing agents such as hydroxylamine hydrochloride added to the t-PA formulations mentioned above The amount of stimulant is 0.1-85 milligrams per kg of body weight, for example 0.1-40 Mix IJ Dalam or 1-85 milligrams into a single mixed preparation.

As absorption enhancer preferably hydroxylamine is used as a non-toxic aqueous salt. Ru. In this case, for example, hydroxylamine hydrochloride instead of hydroxylamine , hydroxylamine bromate, hydroxylamine hydroiodide, hydroxy hydroxylamine sulfate, hydroxylamine nitrate, hydroxylamine acetate and Hydroxylamine propionate is used. The most preferred example is hydroxy Ruamine hydrochloride.

In the present invention, for example, ammonia (ammonium chloride) is used as an absorption enhancer for t-PA. ammonium carbonate and other ammonium salts Ammonium chloride, ammonium acetate, ammonium bromide and ammonium sulfate Urea, mono- and diallylurea such as methylurea, ethylurea, propyl Urea, butyl urea, N, N-dimethyl urea, N, N-diethyl urea, N, N-di Isopropylurea 9-mono and diarylurea e.g. phenyl urea, P-)lyl Urea, N, N-diphecol urea; N, N-dipl, lylurea, thiourea, hydrogen Dantoin, 5-substituted hydantoin e.g. 5-alkyl, 5-aralkyl, 5-substituted hydantoin -Arylhydantoin; 5°5-dialkyl and 5.5-diarylhydantoin Toin examples include 5-methylhydantoin, 5-ethylhydantoin, 5, 5-' ) Methylhydantoin, 5-trimethylenehydantoin, 1,5-tetrame tyrenehydantoin, 5-phenylhydantoin, 5-p-)U-ruhydantoin 5,5-diphenylhydantoin, guanidine, methylguanidine, hydra Zine, alkyl and arylhydrazines such as methylhydrazine, ethylhydrazine Radin, butylhydrazine, phenylhydrazine and diphenylhydrazine; Alkyl and aryl hydroxylamines such as methylhydroxylamine, Ethylhydroxylamine, and phenylhydroxylamine. replacement urine Hydroxylamine, hydrazine, etc. can be used in salt form, e.g. hydrochloride. Ru.

In addition, although t-PA and absorption enhancers are mainly administered simultaneously to the human body, other Administration to salivary mammals such as dogs, cats, livestock and horses is also included within the scope of the invention.

Hydroxylamine, such as hydrochloride, is an inhibitor that is produced naturally in tissue culture. It is known to dissociate A (Levin, Proc, Natl, A cad, Sci, [JSA 806804-6808 (1983):] . Hydroxylamine is also known to inhibit platelet aggregation. l1zuka, chem.

See Pharmacol, Bolt, 20 614-616 (1972) ].

Hydroxylamine also strongly dilates muscles at the injection site, increasing absorption. (Diamond, J., Pharmac.) ol, Exp, Therap, 225.

422-426 (1983)]. These properties were instrumental in the success of this invention.

On the other hand, t-PA and absorption enhancers are usually administered intramuscularly; Administered alone or in combination by subinjection. The reason is hydroxyl The t-PA inhibitor was dissociated from the t-PA (see the Leben reference cited above), and the t-PA inhibitor was injected. enhance the effect of exogenous t-PA or hydroxylamine and thus reduce hemolysis. This is because it reduces the amount of t-PAQ required to perform errands. As mentioned above, Droxylamine is usually administered as a non-toxic salt, preferably the hydrochloride salt. increased absorption The dosage of the promoter, such as hydroxylamine hydrochloride, is within the ranges mentioned above. In the present invention In place of hydroxylamine, other t-PA [dissociation agent of harmful agent can be added] . Absorption enhancer used in injection solutions that are injected intramuscularly by electrical stimulation of the muscle at the injection site It can be used in particular with hydroxylamine hydrochloride. Electrical stimulation is an absorption enhancer of the present invention. Increase absorption of drugs.

As pointed out in the application filed by the same applicant as the applicant, the t-PA muscle Autoinjectors suitable for self-administration can also be used. The following example shows t-PA and hydroxide. Roxylamine hydrochloride can be administered directly intramuscularly with a regular needle and syringe. It was carried out by However, administering drugs with an auto-injector must be done manually. It is thought that the concentration will be higher than the blood concentration obtained by

Rabbits were given intramuscular injections of tPk and hydroxylamine (as hydrochloride), and A treatment method that electrically stimulates the skeletal muscles at the injection site involves administering t-PA to the dog and patient. Equivalent to or better than known methods of causing coronary hemolysis after intravenous injection. It was found that the maximum blood concentration above was obtained. Practical experiments show that after intramuscular injection Facilitating the absorption of t-PA can cause hemolytic effects in coronary arteries and at the same time treat It was found that the necessary blood concentration of t-PA could be obtained.

Because t-PA has limited solubility in normal buffer solutions, Tajo's injection solution was used. . To match dosages, the amount used in rabbits was planned for use in dogs. were chosen to approximate the amount (1 and 1 per injection site in rabbits and dogs, respectively). , using 5 milliliter). Even if he was passionate about the rabbit's muscle mass. However, it was performed as described above. In this case, absorption enhancer per milliliter of injection solution The concentrations were the same for rabbits and dogs. Even if two types Even if the dose of t7PA was administered in proportion to 1 part of the total body weight, the results were compared to dogs. Therefore, in rabbits, hydroxylamine must be administered in extremely large amounts per body weight. Furthermore, the concentration of t-PA in the injection solution will be administered at a 10 times lower concentration. Ru. t-PA'iAm agents, such as thiocyanate, can be used if concentrated to a detectable concentration. It is believed that the volume of injection liquid can be substantially reduced.

In a study on rabbits, t-PA used as described above was found to be effective in obscuring melanoma cells. (1983)] or recombinant DNA technology (Va n　der　Werf.

circulation 6 9 6 0 5-6 1 0 (1984) (r t-PA Genentech Corp,, lot B) 1004 DAX ) produced by.

The results for the two reagents were indistinguishable, so the reagents were combined. buffer Concentration of liquid millimeter per unit time t-P A 0.5 my (0.3M Nacl, 0.0 1% Tween 80.0. OIM potassium phosphate buffer gpH7,s)i was used.

In the dog study, rt-PA (Genentech, lot TE'03 1A) is increased by 20 times using the amine membrane (Am1con) filter system. Concentrated.

DMSO is used internally and in injections as a solution at a concentration of 1% or 3% (V/V). It was. Hydroxylamine hydrochloride t-PA: 43.75mf per liter It was used at a concentration of /. This concentration of hydroxylamine is physiologically well tolerated. Panoramic view of hydrochloride about 13 meters? Comparable to Ag.

rt-PA bath solution to determine the concentration of absorption enhancer that interacts with t-PA. 0.015 50 ng, /+/)k 1% DMS0゜3% DMS0, 175 ~ After adding hydroxylamine (hydrochloride) or DMSO and hydrochloride After the addition of both roxylamine (as the hydrochloride salt), the cells were incubated at 37° C. for 1 hour.

The effect on t-PA has not been determined by radioimmunoassay or functional analysis. I couldn't tell.

56 non-fasted New Zealand white male rabbits weighing approximately 2 kf I researched about it. These animal-derived t-PAs are produced against human) t-PAs. It does not react with the antibodies administered, and it is difficult to measure the blood concentration of exogenously administered t-PA. Does not interfere with radioimmunoassay methods used for Animals are natrium bend parviter Anesthetize the patient with 95% acid at a rate of 21/f'r- through the trachea. The material was aerated. The skeletal muscles at the injection site (inside the landslide) are exposed; Serial blood samples are drawn through the internal femoral vein catheter. injection part To increase blood and lymph flow to skeletal muscles at the 14 volts at 5 pulses per second using a 0.5 inch stainless steel needle, 2.0 Stimulated for m5ec. A single negative bipolar electrode is sufficient. Weight per unit -PAl my f: 15 injections were administered at each of the four sites.

Coronary hemolysis was performed in anesthetized dogs weighing approximately 23% fasted (B ergmann, 5science 220 + 1181-1183].

An occlusive thrompus developed within 5-10 minutes and was confirmed by angiography. series Venous blood samples were made by an indwelling inferior vena cava catheter. at the injection site Electrical stimulation was delivered by three 27 gauge stainless steel needles. That's right One of them serves as a negative control electrode. Parameters used in rabbits It was similar to that. 1. -P A is for one injection site on the exposed sartorius muscle. Direct injection of 1.5 milliliters, total dose 3 milliliters per kilogram of body weight I tried to make it gram. - Also, the total volume of injection solution should be 6 ml total for each dog. It was a lilytuter.

The first experimental end point in the study of 56 rabbits was to determine t-PA activity in the blood. It was sex. The amount of t-PA antibody was determined according to Bergman, Ioc, and Cit. and Van der werL No.

Continuously by the method of Engl, 2Med, 310 609-613 (1984) was analyzed. Functional t-PA activity is determined by Bergman, lot, cit, and Van der werf, No, Engl.

2Med, 310, 609-613 (1984) method. blood test t-PA or solvent alone using a sodium citrate vacutainer tube. 0-4 before injecting meat, immediately after injection, and at selected intervals of 1 minute to 60 minutes thereafter. Blood was collected at ℃.

In actual experiments with dogs, the secondary endpoint was the angiographically suspended crown. This was a hemolytic phenomenon in the arteries. blood pressure, cardiac frequency, electrocardiogram, arterial blood gases and PH, hemoglobin and hemoglobin oxygen saturation were examined.

Two types of experiments investigated possible muscle damage at the injection site. The evaluation was made through a comprehensive survey. Furthermore, a series of blood samples showed that skeletal muscle was Plasma creatine kinase (CK) is significantly released into the circulatory system when injured. In view of the well-known opinion that CK activity was measured by a spectrophotometer, C. in, Cardiovasc.

Res, 7 412-. 418 (1973)].

A series of changes in the blood concentration of t-PA was measured in 56 rabbits, including several groups. It was evaluated. Buffers containing t-PA with or without absorption enhancers alone Buffer containing DMSO with or without hydrochloride. with or without a buffer containing xylamine (as the hydrochloride salt) and with DM with or without buffers containing SO, hydroxylamine (as hydrochloride) and gold. t-PAIl in blood before intramuscular injection of buffer and at intervals after injection. llii analysis.

Electrical stimulation of the muscle at the injection site throughout the blood sampling interval for combinations similar to those above. Tests were conducted with and without the stimulus. Humans that facilitate the absorption of t-PA Once the amount of droxylamine is determined, t-PA and hydroxylamine in the injection solution are determined. To determine the dose-response relationship for the absorption of t-PA as the concentration of min We conducted an experiment. Systematic effect of hydroxylamine on the absorption of t-PA hydroxylamine without t-PA at two injection sites in rabbits. 1 without hydroxylamine when administering and at the other two injection sites. - An experiment was conducted regarding the case of PAI administration.

An experiment was conducted on the acquired appearance determined for rabbits. Blood concentration for treatment The amount of t-PA previously administered to the patient by intravenous injection (0,5-0,75 mφ1 body weight) ) was comparable to f (t - P A 1 my per body weight). t-PA is administered intramuscularly with hydroxylamine (as hydrochloride) within 5-45 minutes. It was. Its administration is generally within 7-10 minutes after insertion of the thrombogenic coil into the blood vessel. This was after taking test records regarding an obstructive blood clot in the lower left anterior coronary artery. . A series of test records were taken at approximately 15 minute intervals. Effect of t-PA on coronary thrombi As a result, the effect of plasma t-PA is correlated with plasma t-PA 9 degrees. dissolve blood clots After bleeding (approximately 15 minutes after t-PA injection), administer pepperin (500 U per body weight). This completely prevented reperfusion. In the absence of foreign activators in the fiber tOm lysis system, Blood clots caused by thrombogenic coronary coils were treated with heparin (n = 40 animals). It remained in spite of the administration of dog). Bonferroni Limited Analysis of variation in researcher's tests on limit values or pairs of data was performed.

Its value is expressed as SE above the mean.

Contains 50 ng of rt-PAO,015 and incubated at 37°C for 1 hour. Hydroxylamine (hydrochloride) (17s1n?Anri, 1% DM with 0.3% DMSO, t or hydroxylamine (hydrochloride) When using SO, there is no appreciable t-PA activity by radioimmunoassay. There was no functionally appreciable t-FA activity.

Concentration of t-PA in blood Prior to intravenous injection of t-PA, t-PA was not present in the plasma of any rabbit. It could not be recognized by radioimmunodetection. None of the four rabbits tested Despite a minor surgical procedure and 60 minutes of electrical stimulation, the patient died. Perceptible outpatient t-PA as analyzed by fibrin plate function test No activity was observed in plasma samples. Human-t-PA is added to the injection solution by exogenous t-PA. - J/'i after using injections with tested solvent combinations without PAi I couldn't recognize it. Administer hydroxylamine hydrochloride to multiple sites to improve overall 262 mV/rrd! 60 from the dog if administered or not administered. There is no t-PA that can be recognized by radioimmunoassay in the plasma sample collected from strawberries. It wasn't there. -]? Contains hydroxylamine hydrochloride in a buffer solution without A Samples were collected every 60 minutes from 4 animals using intramuscular injection sound and electrical stimulation. of dogs (10-53 IU/go-PA) by fibrin plate analysis. No increase in functional activity was observed.

In a comparative study, hydroxylamine hydrochloride alone (262~) was injected intramuscularly into dogs. . The maximum concentration of methemoglobin is 11-13%, and after intramuscular injection (Rho 3) The concentration was reached within 5-15 minutes. Arterial oxygen pressure decreases to a minimum of 93 Hg. Ta. Oxygen saturation in hemoglobin decreased by a minimum of 81%. Also using t-PA Dogs given hydroxylamine hydrochloride without hemodynamic or electrocardiogram No abnormalities were observed.

In the drawing: Figure 1 shows the change in plasma rt-PA degrees (ng/-) over time (minutes). It is a graph. Hydroxylamine hydrochloride 43.75m ~ (Total injection volume - 4ml injected with 2 mg t-P A buffer containing Electrical stimulation was applied to the injection site throughout the sampling period. Immune reactivity and functional activity The activity of t-PA reached its peak immediately after intramuscular injection of an easily absorbed injection solution. This is clear from Figure 1.

Figure 2 shows the hydroxylamine concentration in the injection solution and the radiation immunoassay method. 2 is a graph showing the relationship between the maximum concentration of t-PA in plasma; Injection Jr. The procedure is shown in Figure 1 except that the amount of hydroxylamine hydrochloride is varied as shown. The conditions were the same as those described above.

Figure 3 shows the amount of t-PAO administered by intramuscular injection to six rabbits and the maximum plasma concentration. It is a figure showing the relationship with t-PA activity value. The total amount of t-PA to be administered is illustrated. The weight was removed under the same conditions as in Fig. 1, except for the points changed to . A- is radioimmunoassay (by law) showed appreciable activity. B is amidolytic It showed functional activity. Measured at 1 hour intervals for total time-activity (n-30) The dosage corresponding to the change in time was determined by variable (P(0,001) analysis) It was so important.

Figure 4 shows the case in which t-PA was injected intramuscularly into each of the three rabbits after making it easier to absorb. , is a graph showing temporal changes in t-PA concentration in plasma. The conditions are shown in Figure 1. It was the same as the one I did.

Figure 5 shows blood plasma measured by radioimmunoassay in dogs undergoing coronary trompolysis. This is a graph showing the relationship between a series of changes in t-PA and the passage of time (minutes). . Trompolysis is performed using a trompogen coil inserted into the left anterior lower coronary artery. is triggered at the tip of a coronary catheter. Coronary thrompolysis is t-P It is performed by intramuscularly injecting an injection solution that facilitates the absorption of A.

(Thrombogenic coils cause the formation of blood clots, which are treated with angiographic dyes in peripheral blood vessels.) Unable to fill and close to coil appearing as bright rectangle It was found that the lack of transparency in blood vessels caused the formation of blood clots.

)t-PA’! i: Intramuscular administration (total of 6 ml divided into 4 injection sites) 3 tnry, Ary f: use fr,) for the injection solution 15 minutes after the injection. By electrically stimulating the muscles in the area, the coil is inserted into peripheral blood vessels close to the coil. Hemolysis of the clot was evident by angiography. t-PA activity in plasma is absorbed The maximum value was reached quickly after easy intramuscular administration of t-PAi. Activity is measured by taking a sample. It was maintained throughout the period. The second maximum value is for each of the three dogs tested. It was observed that

Blood concentration of t-PA after intramuscular injection As shown in Table 1, the buffer solution alone was The blood concentration of t-PA increased after several minutes. DMSOe added to injection solution did not increase plasma t-PA concentration. In contrast, hydroxylamine salt t-PA, whose absorption is increased by salt, reaches its maximum blood concentration 5 minutes after injection, and t-PA It was about 40 times higher than when no silamine was used. Determined by radioimmunoassay Note that the series of changes in FIA-active t-PA activity analyzed on fibrin plates An injection solution containing hydroxylamine hydrochloride throughout the injection solution to facilitate absorption of t-PA. It is measured after being injected intramuscularly and applying electrical stimulation to the muscle at the injection site. The results shown in the figure were obtained and confirmed by radioimmunoassay after intramuscular injection of t-PA. Concentration of t-PA in plasma (ng/-) 0 0 ± 0 0 ± 0 0 ± 0 5 8±2 11±4431±521115 9±2 8±2146±16” 30 9±2 9±1 85±17' 6010±3 10±1 53±11'1 Measured values are mean±SE. all The injection solution contained 2q of t~PA in a total of 4- (1- per injection site).

The concentration of hydroxylamine hydrochloride was 43.75 mg/ml. all The experiments in the table were conducted by applying electrical stimulation to the muscle at the site of myocardial infarction.

Qin-p<o, t-Pk in oi buffer alone or in buffer containing DMSO compared.

t1 administered into muscle. ’A absorption is due to increased intramuscular blood flow caused by electrical stimulation. In order to determine whether this can be achieved, an injection containing +-PA in the buffer alone, D Injection solution containing tT,'A in a buffer solution containing MSO, hydroxylamine hydrochloride After each injection solution containing t-PA was injected into the buffer solution that had been added to the entire layer, electrical stimulation was applied. Or experimented without using it. Inject t-PA that does not contain hydroxylamine hydrochloride. The blood concentration of i-PA was small in the case of electric pricking 9 (n = 11 (animals) were not denatured at low concentrations. However, hydroxy Animals treated with t-PAi in combination with Ruamine hydrochloride and electrical stimulation (n-15) In this case, the maximum concentration reached an average of 258±32% regardless of the absorption time of t-PA.

As shown in Figure 2, the maximum blood concentration of 1-PA that can be recognized by radioimmunoassay The degree was proportional to the amount of hydroxylamine hydrochloride in the injection solution. hydroxylamine In an injection solution containing 1% DMSO (hydrochloride) and 3% added, the amount of t-PAO is kept constant. Comparing the results with hydroxylamine hydrochloride alone when Maximum blood concentrations were not increased. Radioimmunity after exogenous administration of t-PA The functionally active t-PA, which can be recognized by the epidemiological assay, is the hydroxyl in the injectable solution. When the amount and concentration of sylamine hydrochloride are kept constant (Figure 3), the range is more than 1 times It was proportional to the concentration of L-PA. As shown in Figure 4, the blood concentration of t-PA rose rapidly and reached its maximum value 4-5 minutes after injection. Can detect t-PA in plasma Concentrations were achieved in each case as quickly as 1 minute after intramuscular injection.

Maximum plasma concentration of t-FA facilitated absorption by hydroxylamine hydrochloride The increase in PH did not occur simply by lowering the PH in the injection solution. two The PH of each injection in the animals was 5.9 in the absence of hydroxylamine.

The t-PA concentration in plasma 5 minutes after injection was only 6nS. Hydroxyl The increase in t-PAfi degree due to amine hydrochloride is simply due to the body of hydroxylamine hydrochloride. It is not based on cyclical effects. Hydroxylamine weakens the muscles in your right thigh. In two animals in which buffer containing t-PA was injected into the left thigh muscle, t-PA was injected into the left thigh muscle. The maximum blood concentration of PA is the concentration in Table 1 when t-PA is injected using buffer alone. It didn't go overboard.

The amount of absorption enhancer per kilogram of body weight used in rabbits was significantly higher than the amount used in dogs. Although this amount is expected to be the maximum amount that can be used clinically, the highly concentrated injection solution Quite high concentrations were used to determine whether it was harmful to muscles or not. cormorant Regarding Sagi K, blood 1cK is surgical procedure, hydroxylamine and t-PA, If electrical stimulation is used at the same time as injection of buffer solution alone, Same concentration compared to the value (696±63 IU/IJ compared to 690±8 There was no significant difference in 2 IU/IJ 30 minutes after injection. dog For hydroxylamine 175 m (J all around or without [-P At the end of the study, plasma CK increased to 18% of baseline when administered A. Ta. No hematoma was seen on cursory observation. Note obtained 2 hours after injection Light microscopic observation of fragments from the injection site revealed only slight interstitial hemorrhage and inflammation. It was only noticeable.

A t-PA injection made easier to absorb with hydroxylamine hydrochloride was administered to rabbits. Post-approximate experiments on dogs to determine whether they cause coronary thrompolysis A preliminary experiment was conducted. with (n-3) or without t-PA. Absorption time of t-PA after intramuscular injection of hydroxylamine hydrochloride (n-3) (average 166/1211IOIIHg) at least 2 minutes after injection without changing the to 144/104AH2). Heart rate is 2 minutes after injection increased temporarily by 32% of the average maximum value. Hydroxylamine for ventricular arrhythmia It didn't happen alone. After intramuscular injection of t-PA (3 mqAy), electricity was applied. Coronary thrompolysis occurred within 15 minutes when the full target stimulation was administered, and this was a cause for concern. It was discovered by ventricular arrhythmia. Similar results were obtained for each of the three animals used in the study. obtained.

Plasma t-PA values followed a similar time course, but values observed in rabbits It was lower. This difference is due to the large absorption of rt-PA in rabbits. and or based on certain differences in the large ratio of injection volume to muscle mass. it is considered as. Furthermore, as shown in Figure 5, 1-PA blood was added to each dog. Circulation or t-PA slow The radioimmunoassay method may cause changes in the lymphatic circulation. The second peak of t-PA that is perceptible occurs approximately 40 minutes after the first peak has disappeared. observed. At the same time, t-PA was gradually released from storage sites in skeletal muscles.

In this case, the blood concentration necessary for treatment of functionally active t-PA may not be obtained and the coronary artery Pulse thrombolysis can be induced by intramuscularly injected substances that facilitate absorption. It turns out that Plasma activity reached its maximum within 5 minutes after injection and then rapidly decreased. Do a little. This is consistent with the known half-life of t-PA in the circulation. in dogs Blood levels of t-PA sufficient to cause coronary thrombolysis are Achieved within 15 minutes despite the presence of arterial thrombogenic film . Absorption of t-PA can be achieved by adding hydroxylamine to the injection solution and by electrical stimulation. This is achieved by increasing blood flow within the skeletal muscles. prominent bones No muscle damage occurred.

Judging from the results of in vitro studies, low blood concentrations of t-PA in plasma are associated with early onset of t-PA. t-P bound to fibrin is suitable for dissolving blood clots in Because the microbiological half-life of A is substantially longer than that of circulating t-PA, Initial coronary trompolysis after initiation of trompolysis was used in the above experiments. A smaller amount of t-P A or hydroxylamine hydrochloride or Id + -P It is believed to be achieved using both A and hydroxylamine hydrochloride. (Brommer+Thromb.

Re534 109-115 (1984); Tran-Thang.

Blood 63 1331 1337 (1984); Bergmann , cir-culation 70 mouths: 108 (Abstract) (1984)].

Reducing injection volume reduces the required dosage of hydroxylamine or other absorption enhancers This also reduces potential damage to the entire muscle at the injection site.

Currently, t-PA and other active agents in undissolved fiber systems are available for direct injection into blood vessels. Therefore, it was only administered. The present invention, as previously explained, medical treatment by a quasi-medical specialist or by a patient who receives instructions over the telephone. To provide an alternative method of t-P A administration that can potentially be modified to promote Ru.

Hydroxylamine is another absorbent that did not give satisfactory results for t-PA. It was used after numerous attempts at yield enhancers. Side effects, i.e. Metahemoglobinemia can be induced by the dosage used and the amount of oxygen to the cells. supply is not restricted. The current concentration of hydroxylamine in the injection solution If we strictly determine the absorption of t-PA by similarly judging from the results, Since the total amount of hydroxylamine required is very low, metahemoglobin The induction of bloodemia can be further reduced by increasing the t-PA concentration. Therefore, even in patients with Ishiemi heart disease, the effect is only slight. Probably not. Metahemoglobinemia associated with the use of absorption enhancers is unacceptable. If the substance is very heavy, use methylene blue or glutathione together. This problem can be minimized if such auxiliary means are used. C.L. ayne, J, Phar-macol, Exp, Therap, 16 536-44 (1969)].

The blood concentration comparable to the blood concentration of t-PA obtained in the present invention is found in experimental animals and coronary artery thrombosis without causing the patient to develop a gross hemolytic state that may lead to bleeding. bring about politics. Plasma t-PA after intramuscular injection of easily absorbed t-PA The time course of the increase is particularly advantageous because the peaks are sharp. Become a quasi-medical specialist Directly after administering self-medication treatment by auto-injector or treatment There are cases where people are placed under medical protection. In this case, total administration of t-PA by intramuscular injection When injected into a vein, anticoagulants or reocclusion are recommended, as the blood concentration decreases rapidly. Initiated with the means of ponding adopted to prevent and during which information on definitive co-severance is obtained.

Myocardial regeneration by facilitating the absorption of t-PA administered by intramuscular injection Perfusion may occur, but arrhythmia may require cardiac catheterization laboratory or coronary intervention. easily controlled by hospitalization in an institution (with no medical protection) There is an insidious danger to the patient. In this case, anti-cardiac or anti-arrhythmic drugs such as Advantageously, the patient is treated with docaine or an alpha-adrenergic blocker.

One of the following methods is preferred to prevent reperfusion or platelet aggregation: I found out.

1. Thromboxane A (thromboxane A2) inhibits thromboxane synthetase. Harmful agents such as 4-(2-CIH-imidazol-1-ylfethoxy)benzoin Inhibitory synthesis with hydrochloride (dacixiben).

2 Thromboxane A (thromboxane A2) (e.g. [1α, 2β (5Z) , 3β(IE), 4α]-7-(3-(3-cyclohexyl-3-hydroxy- 1-propenyl)-7-oxabicycloC2,2,1)hebdo-2-yl]-5 -heptenoic acid) (SQ 27,427) by injecting an antagonist for its receptor. How to do it.

38 Other inhibitors of platelet aggregation such as aspirin, indomethacin, naproxy Method of injecting sulfinpyrazone and sulfinpyrazone.

Examples of agents to prevent reperfusion or platelet aggregation are t-PA and absorption enhancers. For example, it can be administered simultaneously or sequentially with hydroxylamine hydrochloride. reperfusion or Reagents to prevent platelet clotting can be administered in the usual manner, e.g. intramuscularly, intravenously or can be administered orally.

Receptors and antagonists for preventing platelet reperfusion are e.g. Amounts of 10~ can be administered.

FIG.1 Ichima (9) FIG.2 Moan-ma (numerous) FIG.5 international search report 1“””’-’　PCT/US85104503“

Claims (44)

[Claims] 1. In a method of administering t-PA to a mammal, the method includes: t-PA in the blood by administering effective amounts of absorption enhancers substantially simultaneously. How to increase absorption. 2. Item 1 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. Method described. 3. 2. The method according to item 2, wherein the absorption enhancer is hydroxylamine hydrochloride. 4. 2. The method according to item 1 above, wherein the administration is performed by intramuscular injection. 5. 2. The method according to item 2 above, wherein the administration is performed by intramuscular injection. 6. 4. The method according to item 3 above, wherein the administration is performed by intramuscular injection. 7. 2. The method according to item 1, wherein the mammalian animal is a human. 8. 2. The method according to item 2, wherein the mammal is a human. 9. 4. The method according to item 3, wherein the mammal is a human. 10. 5. The method according to item 4, wherein the mammal is a human. 11. 6. The method according to item 5, wherein the mammalian animal is a human. 12. 7. The method according to item 6, wherein the mammal is a human. 13. It involves applying electrical stimulation to the injection site to increase blood flow within the muscle. The method according to item 4 above. 14. It involves applying electrical stimulation to the injection site to increase blood flow within the muscle. The method according to item 5 above. 15. It involves applying electrical stimulation to the injection site to increase blood flow within the muscle. The method according to item 6 above. 16. It involves applying electrical stimulation to the injection site to increase blood flow within the muscle. The method according to item 10 above. 17. It involves applying electrical stimulation to the injection site to increase blood flow within the muscle. 12. The method according to item 11 above. 18. It involves applying electrical stimulation to the injection site to increase blood flow within the muscle. 13. The method according to item 12 above. 19. For intramuscular injection, an effective amount of (1) t-P to enhance absorption of t-PA; A formulation comprising A and (2) a reagent that enhances absorption of t-PA in blood. 20. Item 19 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. The formulation described. 21. 20. The formulation according to item 19, wherein the absorption enhancer is hydroxylamine hydrochloride. 22. t-PA and t-PA absorption enhancer are separated before use. The formulation according to item 19. 23. 23. The formulation according to item 22, wherein the absorption enhancer is hydroxylamine hydrochloride. 24. 24. The formulation according to item 23, wherein the absorption enhancer is hydroxylamine hydrochloride. 25. an effective amount of a t-PA inhibitor dissociating agent to increase the dissociation of the inhibitor from t-PA; The physiological requirements of t-PA include administering t-PA to the mammal. A method of increasing the absorption of t-PA in the blood. 26. 26. The method according to item 25, wherein the inhibitor-dissociating agent is injected into the muscle. 27. Item 25 above, wherein the inhibitor-dissociating agent is hydroxylamine or a non-toxic salt thereof. Method described. 28. 28. The method according to item 27, wherein the inhibitor-dissociating agent is injected intramuscularly. 29. 28. The method according to item 27, wherein the inhibitor-dissociating agent is hydroxylamine hydrochloride. 30. The t-PA inhibitory and dissociative agent is administered without administering exogenous t-PA. The method according to item 25. 31. The t-PA inhibitory and dissociative agent is administered with exogenous t-PA, and the t-PA The amount of the above-mentioned 25. The method described in section. 32. Effective doses of drugs that inhibit blood vessel occlusion or platelet aggregation in the blood 2. The method according to item 1 above, which is administered simultaneously or sequentially. 33. Item 32 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. Method described. 34. 33. The method according to paragraph 32, wherein a trompoxane synthetase inhibitor is used. . 35. Item 34 above, wherein the trompoxane synthetase inhibitor is imidazole. How to put it on. 36. 36. The method according to item 35, wherein the imidazole is dazoxiben. 37. Item 36 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. Method described. 38. Item 34 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. Method described. 39. Item 32 above, wherein an antagonist to the receptor of trompoxane A is used. the method of. 40. 39. The method according to item 39, wherein the antagonist is SQ27,427. 41. Item 40 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. Method described. 42. Item 39 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. Method described. 43. Inhibitors include aspirin, indomethacin, naproxa or sulfinpira 43. The method according to item 42, wherein the method is zonal. 44. Item 43 above, wherein the absorption enhancer is hydroxylamine or a non-toxic salt thereof. Method described.